Tuberculosis (TB), one of the most widespread infectious diseases, is the leading cause of death due to a single infectious agent among adults in the world. Mycobacterium tuberculosis is the most common cause of human TB.
However, an unknown proportion of cases of zoonotic tuberculosis are due to M. bovis with some due to Mycobacterium avium. Thus, infection of the animal population not only places a strain on economic resources but also presents a threat to human health. The requirement for successful diagnostic assays and potential vaccines has increased due to the recent rise in TB levels in the cattle population of countries such as Great Britain and the wild life reservoirs of the world (Hewinson et al 2003).
Great Britain performs some 4.6 million tests on bovine TB, costing the tax payer £88 m per annum (TB Conference M. Bovis IV Dublin 2005). Bovine TB varies regionally within GB, with the worst incidents seen in South Wales, Cornwall and Gloucestershire where 25% of all animals are infected. The incidence of TB is increasing at a rate of 2.5% per year in previously uninfected herds (TB Conference M. Bovis IV Dublin 2005).
In order to monitor and control the disease herd profiling is necessary. However, the methods currently used to monitor tuberculosis in animals suffer from a number of drawbacks. Nowadays, the disease control programmes for bovine TB carried out in most countries (i.e. US, Australia and GB etc.) are based on a test and removal strategy utilizing the intradermal skin test, which relies on PPD, a purified protein derivative of M. bovis strain AN5, to elicit an immune response in infected cattle (Caffrey, 1994; Monaghan et al., 1994).
In cattle, the intradermal skin tests currently used are the Caudal-fold Tuberculin Test (CFT) and the Comparative cervical tuberculin test (CCT). The Caudal-fold Tuberculin Test (CFT) is the Primary screening test used to identify cattle herds potentially infected with bovine tuberculosis. It measures the immune response to Mycobacterium Injecting Purified Protein Derivative (PPD) tuberculin (M. bovis AN5). If the animal's immune system recognizes the PPD, inflammatory cells (white blood cells) migrate to the injected site to help get rid of the foreign material (PPD). This cell mediated immune response may be recognized by swelling or discoloration at the site where PPD was injected. However, in 5% of cases, the CFT test may result in false-positive test results (due to exposure to or infection with other closely related bacteria, such as M. avium and M. paratuberculosis) or, in 15% of cases, in false-negative test results—where a very early stage of infection with bovine TB is not detected. As a follow up test to the CFT, the Comparative cervical tuberculin test (CCT) may be performed. The CCT test is performed in the cervical (neck) region and is a more definitive test designed to determine if a response noted on the CFT test is more likely due to infection with M. avium or M. bovis (injected with PPD avian and PPD bovine). CCT Test-suspect cattle are subjected to further testing using necropsy and further diagnostic testing.
Disease control based on the skin test can be complimented by the gamma-interferon test, which measures the animals T cell response when exposed to PPD material. The gamma-interferon test is utilised as a second line diagnostic for ‘skin test positive’ animals (reports suggest it is more sensitive, but sometimes less specific than skin test). It detects the cell-mediate immune response that develops following M. bovis infection (2 weeks).
The Gamma-Interferon test is not used as a primary test for mass screening on its own because it does not detect all skin test-positive infected animals; it is relatively expensive and is less specific than the CCT.
Due to their modes of action, the specificity of both the intradermal skin test and the gamma interferon test will always be an issue. The cross reactions induced by different mycobacteria strains and environmental mycobacteria such as M. microti and M. africanum and the conflicting requirements between specificity and sensitivity of the test antigens, all accrue to the difficulties in establishing a satisfactory serological protocol for bovine TB.
One of the problems associated with the complex antigenic nature of mycobacteria is the definition of those proteins which are important targets of the immune system and are thus likely present in large numbers of field samples. It is also important to recognize that there is regional variation in the infectious M. bovis strains.
An antibody assay developed using strain specific proteins could resolve both specificity and sensitivity issues.
A number of methods of discriminating between strains of tuberculosis have been suggested. U.S. Pat. No. 6,686,166B, WO 2004/083448A, US2004/0063923A, and U.S. Pat. No. 6,291,190B each describe 129 genetic marker sequences which are suggested for use in the identification of strains of mycobacteria. However, as described herein the mere identification of such markers does not equate with practical utility in a diagnostic method. Many markers are not expressed sufficiently to reliably be used in the identification of a strain. Further, as described further below, the amount of expression of individual markers varies considerably, not only between strains, but within strains geographically and within strains dependent on the stage of infection.
US2005/0272104A suggests the use of the PPD antigens ESAT-6 and CFP-10 in the detection of Mycobacterium tuberculosis in humans. In general however, antibody (Ab) tests based upon PPD tuberculins are characterised by a low discriminating power, with the distribution of the antibody titers between infected and non-infected animals being widely overlapping (Amadori et al. 1998). To date, a diagnostic test which accurately and reliably diagnoses the presence of tuberculosis infection has eluded the field.